Modification of cytosolic, nuclear, and mitochondrial proteins on serine and threonine residues by N-acetylglucosamine (O-GlcNAc) provides a mechanism by which glucose flux rates in the cell can feed back to regulate the function and synthesis of proteins involved in metabolism and growth. The hexosamine biosynthesis pathway (HBP) provides the substrate for this modification, UDP-GlcNAc. At normal to high glucose levels, production of UDP-GlcNAc and subsequent protein modifications are reflective of glucose flux rates, hence the pathway serves a nutrient sensing function. The understanding of this pathway, however, lags seriously behind that of other posttranslational modifications such as phosphorylation for several technical difficulties associated with studying the modification and the enzymes involved in the process. Recently, however, major advances have been made that should allow us to overcome many of these barriers. We propose to take advantage of these advances to address gaps in understanding O-GlcNAc modification by studying the glycogen synthesis pathway. Glycogen synthesis is a critical factor in metabolism and diabetes, but it is incompletely understood despite extensive study. Its regulation, for example, cannot be fully explicated by considering only its classic phosphorylation/dephosphorylation events. Our hypothesis, based on progress in the previous granting period and new preliminary data, is that O-glycosylation of key regulated enzymes in the pathway will affect both their intrinsic activity and their targeting to the glycogen granule. Furthermore, we hypothesize that the degree of glycosylation will be affected by regulation of OGT expression, activity, and targeting. Our overall strategy will be to make the initial surveys in cultured HepG2 cells and then test for relevance in mouse models. We propose to: 1. Define the effects of hexosamine flux on all key enzymes involved in glycogen synthesis. 2. Define the effects of O-GlcNAc at specific sites on glycogen synthase activity and targeting. 3. Determine the degree of specificity of OGT targeting in low vs. high HBP. PUBLIC HEALTH RELEVANCE: We are studying a pathway that is poorly understood but nevertheless has relevance to the mechanisms by which tissues sense and respond to their nutrient status. This sensing mechanism has broad relevance to diabetes, cancer and development. The specific process that we are investigating is how the pathway regulated the storage of glucose as glycogen, an important aspect of regulating blood glucose levels